This proposal addresses the role of the BH3-only protein, PUMA, in the regulation of apoptosis. PUMA is transcriptionally activated by p53 in response to genotoxic stress and is the primary mediator of p53-dependent apoptosis. Thus, the proposed studies into molecular basis of PUMA's pro-apoptotic function are central to the major tumor suppressor pathway in humans. At St. Jude, we collaborate with Doug Green's lab, which discovered the PUMABCL-xLp53BAX apoptosis regulatory system, and Gerry Zambetti's lab, which discovered PUMA as a p53-induced, pro-apoptotic protein. Our preliminary studies have clarified the mechanism of PUMA-induced apoptosis. Our results show that PUMA alone is insufficient to directly promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Instead, PUMA is a de-repressor BH3-only protein and as such cannot directly initiate BAX and/or BAK activation. Instead, PUMA acts indirectly by displacing direct activator proteins (i.e., BID, BIM, and p53) from BCL-xL. Importantly, these results demonstrate that interactions between PUMA and BCL-xL are central to the mechanism of PUMA-induced MOMP and apoptosis. Apart from its nuclear role as a transcription factor, p53 regulates apoptosis in the cytoplasm through interactions with the BCL-2 family of proteins, serving as a direct activator of apoptosis. By default, cytosolic p53 is sequestered in inactive complexes by BCL-xL. However, when expressed, PUMA binds BCL-xL and, through a unique and previously unknown mechanism, releases sequestered p53. Released p53, in turn, directly interacts with BAX, triggering a cascade involving BAX oligomerization, MOMP, release of cytochrome c, and, ultimately, apoptosis. Importantly, PUMA is the only de-repressor BH3-only protein that releases p53 from BCL-xL, unleashing p53's full apoptotic potential. We used biochemical, biophysical and structural methods, as well as functional assays, to discover the mechanism by which PUMA uniquely releases p53 from BCL-xL. In the proposed studies, we aim to elaborate and refine our preliminary understanding of this critical tumor suppressor mechanism. Specifically, our aims are: 1) To elucidate the molecular details of PUMA-induced structural rearrangement of BCL-xL and to fully validate the biological role of this mechanism in p53-mediated apoptosis, and 2) To elucidate the physical basis for interactions between p53 and BCL-xL and how these interactions are disrupted by PUMA binding to BCL-xL. The knowledge gained through the proposed studies will define the specificity of apoptotic signaling mediated by PUMA, BCL-xL and p53. PUBLIC HEALTH RELEVANCE: Understanding the roles of p53 in suppressing tumors is essential for successfully treating cancer patients. The non-transcriptional apoptogenic role of p53 has now been established as an essential component of its tumor suppressor function in response to oncogenic and genotoxic processes. Our proposed studies of PUMA will be key in understanding the mechanism(s) through which both the transcriptional and non-transcriptional apoptogenic roles of p53 collaborate in preventing and maintaining a tumor-free state in humans.